Neuromuscular study of early branching Diuronotus aspetos () yields insights into the evolution of organs systems in Gastrotricha

Bekkouche, Nicolas Tarik; Worsaae, Katrine

Published in: Zoological Letters

DOI: 10.1186/s40851-016-0054-3

Publication date: 2016

Document version Publisher's PDF, also known as Version of record

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Citation for published version (APA): Bekkouche, N. T., & Worsaae, K. (2016). Neuromuscular study of early branching Diuronotus aspetos (Paucitubulatina) yields insights into the evolution of organs systems in Gastrotricha. Zoological Letters, 2, [21]. https://doi.org/10.1186/s40851-016-0054-3

Download date: 08. apr.. 2020 Bekkouche and Worsaae Zoological Letters (2016) 2:21 DOI 10.1186/s40851-016-0054-3

RESEARCH ARTICLE Open Access Neuromuscular study of early branching Diuronotus aspetos (Paucitubulatina) yields insights into the evolution of organs systems in Gastrotricha Nicolas Bekkouche and Katrine Worsaae*

Abstract Background: Diuronotus is one of the most recently described genera of Paucitubulatina, one of the three major clades in Gastrotricha. Its morphology suggests that Diuronotus is an early branch of Paucitubulatina, making it a key taxon for understanding the evolution of this morphologically understudied group. Here we test its phylogenetic position employing molecular data, and provide detailed descriptions of the muscular, nervous, and ciliary systems of Diuronotus aspetos, using immunohistochemistry and confocal laser scanning microscopy. Results: We confirm the proposed position of D. aspetos within Muselliferidae, and find this family to be the sister group to Xenotrichulidae. The muscular system, revealed by F-actin staining, shows a simple, but unique organization of the trunk musculature with a reduction to three pairs of longitudinal muscles and addition of up to five pairs of dorso- ventral muscles, versus the six longitudinal and two dorso-ventral pairs found in most Paucitubulatina. Using acetylated α-tubulin immunoreactivity, we describe the pharynx in detail, including new nervous structures, two pairs of sensory cilia, and a unique canal system. The central , as revealed by immunohistochemistry, shows the general pattern of Gastrotricha having a bilobed brain and a pair of ventro-longitudinal nerve cords. However, in addition are found an anterior nerve ring, several anterior longitudinal nerves, and four ventral commissures (pharyngeal, trunk, pre-anal, and terminal). Two pairs of protonephridia are documented, while other Paucitubulatina have one. Moreover, the precise arrangement of multiciliated cells is unraveled, yielding a pattern of possibly systematic importance. Conclusion: Several neural structures of Diuronotus resemble those found in Xenotrichula (Xenotrichulidae) and may constitute new apomorphies of Paucitubulatina, or even Gastrotricha. In order to test these new evolutionary hypotheses, comparable morphological data from other understudied branches and a better resolution of the basal nodes of the gastrotrich phylogeny are warranted. Nonetheless, the present study offers new insights into the evolution of organ systems and systematic importance of so-far neglected characters in Gastrotricha. Keywords: Neurobiology, Nervous system, Musculature, Ciliary structures, Meiofauna, , , Molecular phylogeny

Background kinorhynchs (Nematorhyncha [5]) or Gnathostomulida Gastrotricha are small, often sub-millimetre, interstitial (Neotrichozoa [6, 7]). Later, molecular phylogenies placed worms, ubiquitously found in most aquatic environments them within Spiralia with uncertain affinities; within the with a long debated phylogenetic position [1–3]. They debated group , comprising Gastrotricha, Platy- were first considered closely related to various meiofaunal, helminthes and Gnathifera [1, 8, 9]. Recent phylogenomic groups, such as rotifers (Trochelminthes [4]), studies propose a sister group relationship between Platyhelminthes and Gastrotricha [3, 10]. However, the * Correspondence: [email protected] controversy over the phylogenetic position of Gastro- Marine Biological Section, Department of Biology, University of Copenhagen, tricha masks other problems within the group. Indeed, Universitetsparken 4, 2100 Copenhagen Ø, Denmark

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 2 of 29

compared to the diversity and omnipresence of these synapomorphy [34]. These recent reports were used to , relatively few phylogenetic and detailed mor- infer the plesiomorphic arrangement of the musculature phological studies have been conducted on this group of Gastrotricha as constituted by two ventro-lateral longi- and the evolution of, e.g., nervous system, muscular sys- tudinal muscles surrounded by outer circular muscles, tem and nephridia is unresolved [11–13]. Its diversity and longitudinal splanchnic muscles surrounded by also remains largely unexplored, as exemplified by the helicoidal and intestinal circular muscles [2]. In Paucitubu- recent erection of the family Hummondasyidae (Macro- latina, the longitudinal muscles appear to be more numer- dasyida) in 2014 [14] and the genera Thaidasys in 2015 ous, and the outer circular muscles, if present, are [15] and Bifidochaetus in 2016 [16]. incomplete and consist of dorso-ventral muscles [2]. These Diuronotus [17] is another recently described gastro- dorso-ventral or semi-circular muscles are found in marine trich (2005), comprising two described species: chaetonotids [22], but are often missing or highly reduced Diuronotus aspetos Todaro et al. [17] and, Diuronotus in freshwater chaetonotids [11, 22, 33] emphasizing the im- rupperti Todaro et al. [17], and one undescribed species portance of studying the marine Diuronotus in order to re- Diuronotus sp. [18–20], transferred from Halichaetonotus solve their evolution and contribute to the broader [17]. These are all found in marine interstitial environments understanding of muscular evolution within Gastrotricha. of the North Atlantic; D. aspetos from Greenland [17] and To date, only one confocal study on Xenotrichula has Germany [2, 21], D. rupperti from Denmark [17] and Diur- described the nervous system of a member of Paucitubu- onotus sp. from North Carolina, USA [18]. Diuronotus was latina in detail [12], while it has been extensively described placed in Muselliferidae (Paucitubulatina, Chaetonitida) for Multitubulatina (Neodasys) [13] and in several Macro- next to Musellifer [22] with which it shares the presence of dasyida with combined immunohistochemistry and CLSM a ciliated so-called ‘muzzle’ (or snout) and specific ultra- (e.g., [35, 36]), or transmission electron microscopy structural traits of scales and sperm [23]. (TEM) [37]. One of the conclusions of the Xenotrichula Gastrotricha are divided into two main taxa: the sup- study [13] is the low structural variation of the nervous posedly monophyletic Macrodasyida and the possibly system within Gastrotricha, which always comprises a bi- paraphyletic Chaetonotida, divided further into the Mul- lobed brain with a ventral commissure, a pair of anteriorly titubulatina, (consisting of one genus, Neodasys, and projecting longitudinal nerves, a pair of ventro-lateral possessing multiple adhesive glands) and the diverse nerve cords along the trunk, and a terminal commissure. Paucitubulatina (possessing generally only two adhesive These features were also interpreted as ancestral condi- tubes) [24]. Muselliferidae, belonging to Paucitubulatina, tions of Gastrotricha in Kieneke and Schmidt-Rhaesa is the possible sister group to all remaining Paucitubula- (2015) [2, 12]. Yet only a single Paucitubulatina, Xenotri- tina according to morphological [22, 25] and molecular chula, was considered for this state reconstruction. More- [26, 27] studies. However, Paps and Riutorts [28] over, substantial variation exists, such as the presence of propose an alternative topology in which Xenotrichuli- an additional ventral nerve in Oregodasys cirratus Rothe dae is positioned as sister group of the remaining Pauci- & Schmidt-Rhaesa [38] and dorsal nerves in Xenodasys tubulatina, and Muselliferidae being the sister group to riedli (Schöpfer-Sterrer, 1969) [39, 40], or additional trunk . Kieneke et al. [29] find Proichthydiidae commissures in Dactylopodola and Oregodasys cirratus. as sister group to the remaining Paucitubulatina, with These studies highlight the unexplored diversity of gastro- Muselliferidae forming a clade together with Xenotrichuli- trich nervous systems, which may be especially relevant in dae sister group to other Paucitubulatina. These different the diverse group of Paucitubulatina, in which only one topologies overall suggest a key position of Muselliferidae study of the nervous system has been reported so far [13] within Gastrotricha, emphasizing the importance of this and given the total lack of data on Muselliferidae. family for understanding the evolution of Gastrotricha. In- Several studies have described the ultrastructure and deed, some features of Muselliferidae, namely its marine repartition of protonephridia in Gastrotricha, with a few habitat and well-developed hermaphroditism, are thought of them addressing species of Paucitubulatina [41, 42]. It to be plesiomorphic character traits of Chaetonotida. has been suggested that members of Paucitubulatina However, detailed morphological studies on this family always possess one pair of trunk protonephridia [41], al- are lacking, most likely due to the paucity of these animals though again, data on Muselliferidae are lacking. Each and their late discovery [26, 30]. nephridium was found to encompass two monociliated Recently, a series of papers employing confocal laser terminal cells with coaxial cilia, a long canal cell, and a scanning microscopy (CLSM) described the detailed mus- nephridopore cell [2]. cular arrangement of several Paucitubulatina, namely In order to enhance our understanding of the evolution Musellifer [22], Xenotrichulidae [22, 31], Chaetonotidae of major organ systems within Gastrotricha we acquired [22, 32], and [11, 33], and notably, the new morphological data on Diuronotus aspetos,using helicoidal musculature, proposed to be a gastrotrich CLSM techniques and immunohistochemistry to describe Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 3 of 29

the detailed arrangement of the musculature, nervous sys- The 28S primer set used was 28SD3/28SG758 [45, 46] tem, and ciliation. To assess the previously proposed rela- with an annealing temperature of 53 °C. tionship of D. aspetos within Muselliferidae, we analyzed All newly generated and integrated sequences were de- the phylogenetic position within Chaetonotida, using mo- posited in the GenBank ® database with the following ac- lecular data. In this phylogenetic context, the morphology cession numbers KX531001 to KX531007 (Table 1). of Diuronotus is compared and discussed relative to other Chaetonotida, and Gastrotricha in general, leading to the Phylogenetic analysis proposition of several new homologies. Sequences were cleaned on BioEdit [47], and a consen- sus has been realized from the reverse and forward Methods sequences. Sequences were blasted on NCBI [48]. In Collection parallel, COI, 18S and 28S of Diuronotus aspetos were For Diuronotus aspetos, the samples were taken with a mini found from its transcriptome [3], using Blastall from van Veen grab from shallow water (3–6mwaterdepth)of NCBI. Sangers and transcriptome acquired COI, 18S Flakkerhuk (69°38.63’N 51°51.13’W), Disko Island, West and 28S genes were aligned and compared, showing low Greenland. All specimens were collected during the Arctic quality and short length of Sangers sequences. Conse- summer in August 2013. Sediment was well-sorted sand of quently, COI and 28S of the transcriptome were kept, fine to medium grain size. The specimens have been while a consensus of 18S from the transcriptome and extracted with MgCl2 narcotization and decantation. the Sangers sequencing was done having an identical For DNA, specimens of Xenotrichula sp. have been overlapping segment. This hybrid approach was possible sampled in Ystad, Sweden (55°26.28’N13°55.44’E) in sub- since the specimens used for the transcriptome and the terranean environments on a beach with fine to medium- Sanger sequences came from the same sample. Se- sized sand, and extracted with MgCl2 narcotization and quences of sp. and Xenotrichula sp. were decantation. Marine Aspidiophorus sp. were sampled from added to the dataset. Sequences of other gastrotrichs ac- cultures of Dinophilus gyrociliatus from Copenhagen Uni- quired from GenBank, based on the tree proposed by versity, where they are contaminants and unfortunately of Kånneby et al. 2012 [49] were added, selecting sequences unknown origin. from each genus (except Bifidochaetus [16]) for which sequences were not available at the time of the analysis), Sequence acquisition and representing the shortest and deepest branches pos- Total genomic DNA was obtained from whole specimens sible. Sequences from Kånneby et al. [26] for Musellifer, using the Qiagen DNeasy Blood & Tissue Kit (Qiagen Inc., Kånneby and Todaro [50] for Neogosseidae, and Todaro Valencia, CA, USA) following the manufacturer’s protocol, et al. [51] for the macrodasyidans, outgroups have add- except for performing the DNA elution in 160 μLofAE itionally been collected from GenBank. Subsequently, buffer in order to increase the final DNA concentration. the sequences were aligned gene per gene with Muscle Polymerase chain reactions (PCR) using Sanger based in Seaview [47], checked by hand, and the three genes markers were prepared to a final volume of 25 μLwith were concatenated with Sequence Matrix [52]. Finally, 12.5 μL of GoTaq® Green Master Mix (Promega Corpor- this dataset was analyzed with Bayesian inference in ation, Madison, WI, USA), 1 μLofeachprimer(10μM MrBayes 3.2.6 [53] under the model GTR + I + Γ. The concentration), 10–8.5 μL of Milli-Q water (adjusted to gamma shape parameter, the substitution rates, the pro- amount of DNA template), and 0.5–2 μL of DNA tem- portion of invariable sites, and the character state fre- plate. Reaction mixtures were heated in a Bio-Rad G1000 quencies were all unlinked. The dataset was partitioned Thermal Cycler at 94 °C for three minutes, followed by according to each gene and by codon position for COI 35–40 cycles (primer specific) of 94 °C for 30 s, specific and analyzed with four MCMC chains for each run, for primer pair annealing temperatures for 30 s, and an exten- 30,000,000 generations. Chains were sampled every sion at 68 °C for 45 s (unless indicated otherwise), and a 1000th generations and the burn-in was set to 25 %. final extension phase of 5 min at 72 °C. The COI primer Convergence of the two runs as well as analysis quality set dgLCO1490/dgHCO2198 [43] was run with two cyc- was ascertained by checking the log likelihood graphs, ling steps, both variable in temperature. COI annealing the average standard deviation of split frequencies, and temperatures were 45 °C for 45 s and 51 °C for 45 s, re- the model fit with Tracer [54]. spectively with extensions of 30s. Overlapping fragments of the small 18S rDNA (ca. 1800 bp) were obtained using Immunohistochemistry and CLSM paired primers corresponding to fragments 1 and 3 of the Specimens were anesthetized with isotonic magnesium 18S rDNA [44]: (1) 18S1f/18S5R (ca. 900 bp) and (3) chloride and fixed in 3.7 % paraformaldehyde in phos- 18Sa2.0/18S9r (ca. 800 bp) both overlapping. Both primer phate buffered saline (PBS) for 1 to 2 h at room sets (1) and (3) had annealing temperatures set to 49 °C. temperature (RT), followed by six rinses in PBS and Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 4 of 29

Table 1 Sequences used for the phylogenetic reconstruction with their corresponding GenBank accession number. See material and methods for further information on the acquisitions of the sequences of Diuronotus aspetos Species name 18S 28S COI Arenotus strixinoi JQ798537.1 JQ798608.1 JQ798677.1 Aspidiophorus kw654 KX531002 KX531001 No Aspidiophorus ophiodermus JN185463.1 JN185510.1 JN185544.1 Aspidiophorus paramediterraneus JQ798538.1 JQ798609.1 JQ798678.1 Aspidiophorus polystictos TK76 JQ798598.1 JQ798665.1 JQ798727.1 Aspidiophorus polystictos TK75 JQ798597.1 JQ798664.1 JQ798726.1 Aspidiophorus sp.3 JQ798559.1 JQ798629.1 JQ798694.1 Aspidiophorus tentaculatus TK120 JQ798553.1 JQ798625.1 JQ798690.1 Aspidiophorus tentaculatus TK228 JQ798591.1 JQ798659.1 JQ798721.1 Aspidiophorus tetrachaetus JN185505.1 JN185540.1 JN185576.1 laroides JQ798580.1 No JQ798712.1 Chaetonotus cf. sphagnophilus JQ798604.1 JQ798671.1 JQ798733.1 Chaetonotus cf. dispar JQ798561.1 JQ798631.1 JQ798696.1 Chaetonotus cf. hystrix JQ798603.1 Q798670.1 JQ798732.1 Chaetonotus cf. laroides TK86 JQ798602.1 JQ798669.1 JQ798731.1 Chaetonotus cf. maximus TK186 JQ798574.1 JQ798646.1 JQ798706.1 Chaetonotus heterocanthus TK100 JQ798543.1 JQ798615.1 JQ798681.1 Chaetonotus mariae JQ798558.1 JQ798628.1 No Chaetonotus neptuni MT61 JQ798539.1 JQ798610.1 JQ798679.1 Chaetonotus uncinus JQ798540.1 JQ798611.1 No Chaetonotus cf. novenarius JQ798566.1 JQ798636.1 JQ798699.1 Dactylopodola mesotyphle JF357651.1 JF357699.1 JF432036.1 Dasydytes papaveroi TK157 JQ798571.1 JQ798640.1 JQ798703.1 Diuronotus aspetos KX531005 and SRX1121926 KX531006 or SRX1121926 KX531007 or SRX1121926 Draculiciteria tesellata MT63 JN185457.1 JN185506.1 JN185541.1 Draculiciteria tesellata TK142 JN185470.1 JN185516.1 JN185549.1 Halichaetonotus aculifer JQ798550.1 JQ798622.1 JQ798688.1 Halichaetonotus euromarinus JQ798551.1 JQ798623.1 No Haltidytes squamosus JQ798567.1 JQ798637.1 No macrops JN185469.1 JN185515.1 JN185548.1 Heterolepidoderma sp.2 JN185485.1 JQ798644.1 JN185563.1 Heteroxenotrichula squamosa JQ798542.1 JQ798613.1 No skandicum TK182 JQ798573.1 JQ798645.1 JQ798705.1 Ichthydium squamigerum JQ798607.1 JQ798674.1 JQ798736.1 Kijanebalola devestiva TK240 KR822112.1 KR822117.1 KR822120.1 Lepidochaetus brasilense TK223 JN185495.1 JQ798658.1 JN185568.1 Lepidochaetus zelinkai TK94 JN185503.1 JN185538.1 JN185574.1 squamata TK97 JN185504.1 JN185539.1 JN185575.1 Macrodasys sp.1 JF357654.1 JF357702.1 JF432040.1 Megadasys sp.1 JF357656.1 JF357704.1 JF432042.1 Musellifer delamarei AM231775.1 No No Musellifer reichardti KF578503.1 No No Neodasys chaetonotoideus JQ798535.1 No JQ798675.1 Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 5 of 29

Table 1 Sequences used for the phylogenetic reconstruction with their corresponding GenBank accession number. See material and methods for further information on the acquisitions of the sequences of Diuronotus aspetos (Continued) Neodasys uchidai JQ798536.1 No JQ798676.1 Neogossea acanthocolla KR822114.1 KR822119.1 KR822121 Neogossea antennigera TK232 KR822110.1 KR822115.1 No Ornamentula paraensis TK147 JQ798562.1 JQ798632.1 JQ798697.1 Polymerurus nodicaudus TK165 JN185502.1 JN185537.1 JN185573.1 Polymerurus rhomboides TK217 JN185493.1 JN185533.1 JN185567.1 Stylochaeta fusiformis JN185471.1 JN185517.1 JN185550.1 Xenotrichula cf. intermedia JN185461.1 No No Xenotrichula intermedia MT71 JF357664.1 JF357712.1 No Xenotrichula sp. kw655 KX531003 KX531004 No Xenotrichula punctata JN185464.1 JN185511.1 No Xenotrichula sp. TK121 JF970234.1 No No Xenotrichula sp.1 JN185466.1 No JN185545.1 Xenotrichula velox TK202 JN185488.1 JQ798652 No Xenotrichula velox TK43 JN185499.1 No No

storage in PBS with 0.05 % NaN3. Triple or quadruple ASSOCIATES, Inc., Alabama, USA) or Vectashield with stainings were performed for the investigation of muscu- DAPI (VECTOR LABORATORIES, Burlingame, USA). lar, nervous, glandular and ciliary systems, including F- The specificity of the antibodies was tested by examining actin staining (Alexa Fluor 488-labelled phalloidin, INVI- specimens, where either the primary or secondary anti- TROGEN, Carlsbad, USA), DNA-staining (405 nm fluores- bodies were omitted. Chicken anti acetylated α-tubulin cent DAPI) and antibodies against neurotransmitters and staining did not produce satisfying results and is therefore tubulinergic elements (monoclonal mouse anti-acetylated not shown in this study (Sigma SAB3500023-100UG). α-tubulin (SIGMA T6793, St. Louis, USA), polyclonal The mounted specimens were scanned using an Olym- chicken anti acetylated α-tubulin (SAB3500023-100UG), pus Fluoview FV-1000 confocal laser scanning microscope polyclonal rabbit anti-serotonin (5-HT, SIGMA S5545) (of K. Worsaae, University of Copenhagen, Denmark), and anti-FMRF-amide (IMMUNOSTAR 20091, Hud- with the acquired z-stacks of scans being either projected son, USA)). Prior to adding the primary antibody-mix, into 2D-images or analyzed three-dimensionally using the samples were pre-incubated with 1 % PTA (PBS + IMARIS 7.1 (BITPLANE SCIENTIFIC SOFTWARE, Zür- 1%Triton-X,0.05%NaN3,0.25%BSA,and5%su- ich, Switzerland). This software package was also used to crose) for 1 h. Samples were incubated over night at RT conduct the measurements presented in the following text in primary antibodies mixed 1:1 with glycerol (in a final according to the conventions introduced by Hummon et 1:400 concentration). Subsequently, specimens were al. [55], i.e. position in the body is given in units (U) as a rinsed in PBS six times and incubated with the second- relative measurements to total body length, measured ary antibodies conjugated with fluorochromes over from anterior to posterior. Schematic hand drawings and night at RT (mixed 1:1 with glycerol; 1:400 goat anti- plate setup were done with Adobe Illustrator CS6 and mouse labeled with CY5 (JACKSON IMMUNO- image adjustments conducted in Adobe Photoshop CS6. RESEARCH, West Grove, USA, 115-175-062), 1:400 goat anti-mouse labeled with TRITC (JACKSON Results IMMUNO-RESEARCH, West Grove, USA, 115-175- Phylogeny 062), 1:400 goat anti-rabbit labeled with TRITC The tree (Fig. 1) shows a fully supported sister group rela- (SIGMA T5268), and 1:200 goat anti-chicken labeled tionship (100 % posterior probability (PP)) between the with Dylight (JACKSON IMMUNO-RESEARCH, West monophyletic Muselliferidae (100 % PP) and Xenotrichuli- Grove, USA, 103-495-1550)). They were rinsed in PBS dae (100 % PP), herein collectively called “group A”.Within five times and one time in 1 % PTA and pre-incubated Muselliferidae the genus Musellifer (100 % PP) (represented for 60 min in Alexa Fluor 488-labeled phalloidin only by Musellifer delamarei (Renaud-Mornant [56]) and (0.33 M in 1 % PTA). Thereafter, specimens were rinsed Musellifer reichardti Kånneby et al. [26]) is found to be the in PBS (without NaN3) and mounted in Fluoromount- sister group to Diuronotus aspetos. Group A is found next G with DAPI (SOUTHERN BIOTECHNOLOGY to “group B”, together constituting the Paucitubulatina Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 6 of 29

Fig. 1 Phylogenetic position of Diuronotus aspetos inferred from Bayesian analysis of 18S, 28S, and COI. This tree is commented in the results section. The analysis includes 58 taxa representing all available genera of Chaetonotida for molecular data on NCBI, and three Macrodasyida as outgroups. Numbers at the nodes represent posterior probabilities in percentages. The picture on the lower left corner is a light micrograph of a live specimen of Diuronotus aspetos Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 7 of 29

(100 % PP), with group B showing a monophyletic Dasydy- body longitudinal muscles mentioned above. Two sets of tidae (100 % PP) and Neogosseidae (100 % PP) nested muscles are strictly limited to thepharyngealregion:apair within “Chaetonotidae”, the latter hereby becoming para- oflateralandapairofdorsalmuscles.Thelateral phyletic. Supports are high in all nodes of the tree, except pharyngeal longitudinal muscle (lplm, Figs. 2d, e and 3c, d) for some of the numerous inner nodes of group B. extends adjacent to the pharynx along its entire length. The pharyngeal dorsal longitudinal muscles (pdlm, Figs. 2d, e, Musculature m, o and 3b-d) extend close to the pharyngeal midline The body wall musculature consists of several pairs of along its total length, ventral to the dorsal longitudinal longitudinal muscles, numerous dorso-ventral muscles, a muscles. Moreover, several somatic and splanchnic longitu- thin helicoidal musculature, semi-circular and complete dinal muscles supply the pharyngeal region. circular muscles, as well as pharyngeal musculature. The The paired ventral longitudinal muscle (vlm, Figs. 2 pharyngeal musculature is especially dense and has an and 3) originating in the head splits along the pharynx organization typical of chaetonotid gastrotrichs, as de- into a complex pattern (see Fig. 3b). One of its branches scribed below in more detail (Figs. 2 and 3). extends more laterally and splits into several sub- branches, supplying the lateral sides of the head. Radial muscles The paired ventro-lateral longitudinal muscle (vllm, The pharynx, sensu stricto, is formed by three rows of Figs. 2 and 3) lines the pharynx until reaching the head, dense radial pharyngeal muscles (rpm, Figs. 2d, e, m and where it bifurcates at U7, one branch extending ventro- 3c, d), and extend to U26 (units are calculated as length laterally and the other dorso-laterally. Each of them sub- from anterior end, relative to total length, see material sequently splits into several minor branches, supplying and methods). The radial muscles are cross-striated and the lateral sides of the head. These muscles together each of them presents three to six Z-discs, which are less with the antero-lateral branch of the ventral longitudinal numerous anteriorly. The myoepithelial nuclei of the muscle (vlm), and the head diagonal muscle (hdm, see pharynx have a distinctive folded and elongated shape below) all supply the antero-lateral part of the head, and (mn, Fig. 2m). The pattern and repartition of these nu- in addition to anchoring the longitudinal muscles for clei seems to be specific and corresponding nuclei could overall body contraction, they may function separately in be found in the same position in different specimens. contraction of the head (Figs. 2j and 3a, c). The paired dorsal longitudinal muscle (pdlm) spans Helicoidal muscles the anterior-most extremity of the pharynx. Helicoidal muscles (hm, Figs. 2n and 3a, b, d, e) are very thin (0.5–1.2 μm) and limited to the anterior half of the Trunk region Three main longitudinal muscles, i.e. ven- specimen. It is difficult to confirm the presence of the tral, lateral and dorsal, are supplying the trunk. The paths helicoidal muscles closest to the pharynx due to the strong of the lateral and dorsal longitudinal muscles are relatively signal of other pharyngeal muscles (dashed lines in straight throughout the body. However, just posterior to Fig. 3c-d). In a few locations along the midline of the the pharynx, the dorsal longitudinal muscle lines the intes- pharynx very faint diagonal fibers were observed, suggest- tine (Figs. 2f and 3c, d, e), while it runs closer to the dorsal ing that the helicoidal musculature is present along the body wall more posteriorly (Figs. 2g, h and 3f, g). The entire pharyngeal region. Distinct helicoidal muscles are ventral longitudinal muscle splits into three muscle bundles found extending from the midgut/pharynx junction at at the anterior trunk. Two of these branches run in parallel U26 until U42, enveloping the dorsal longitudinal muscle, mid-ventrally along the trunk (lvlm, mvlm, Figs. 2a, c, f-h, p but not the ventral or ventro-lateral longitudinal muscles. and 3a, b, f-h), whereas the third branch extends dorso- laterally and supplies the dorso-lateral sides of the body Longitudinal musculature until meeting the median-most branch at U86 (dvlm, Three longitudinal muscles span the entire body length: Figs. 2a, g, l and 3a, b, f). a pair of ventral longitudinal muscles (vlm, Figs. 2 and 3), a pair of ventro-lateral longitudinal muscles (vllm, Figs. 2 and 3), and a pair of dorsal longitudinal muscles Posterior region The median branch of the longitudinal (dlm, Figs. 2 and 3). The ventral longitudinal muscle ventral muscle splits posteriorly into two bundles at U86. bundle splits several times in a pattern described below One very short (8 μm) portion medially supplies the pos- for the different body regions. terior part of the adhesive gland of the posterior tube, while another longer branch extends into the primary Pharyngeal region Several longitudinal muscles are tube, supplying it for approximately two-thirds of its present along the pharynx.Somearelimitedtothe length to U97 (Fig. 3a). Additionally, the lateral branches pharyngeal region, while others are the continuity of the of the ventral and lateral longitudinal muscles also extend Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 8 of 29

Fig. 2 (See legend on next page.) Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 9 of 29

(See figure on previous page.) Fig. 2 CLSM of phalloidin stained muscle of Diuronotus aspetos. Anterior of the specimen is pointing left for a, b and j–p, and dorsal is pointing at the top for d–i. a–n Muscles in green, nuclei in cyan a Ventral view of the maximum intensity projection (MIP) of the whole specimen. b Dorsal MIP of the pharynx. c Dorsal MIP of the posterior specimen. d–i CLSM virtual transverse section of various parts of the specimen: d head, e posterior part of the pharynx, f anterior of the trunk, g posterior of the trunk, h post-anal region of the trunk, i and furca before bifurcation of the tubes. j Dorsal MIP of a sub-stack showing details on the head musculature. k Dorsal MIP of a sub-stack showing details of the furca separation. l Ventral MIP of a sub-stack showing details of the semicircular musculature. m Single section showing details of the inner pharynx. n Dorsal MIP of a substack showing details of the helicoidal musculature. o and p, isosurface reconstruction of the pharynx. o Dorsal view, p ventral view. ag adhesive gland, agn adhesive gland nucleus, aps anterior pharyngeal sphincter, cmag circular muscle of the adhesive gland, dlm dorsal longitudinal muscle, dvlm dorsal projection of the ventral longitudinal muscle, dvm dorso-ventral muscle, hdm head diagonal muscle, hm helicoidal muscles, lplm lateral pharyngeal longitudinal muscle, lvlm Lateral extension of the ventral longitudinal muscle, mn myocyte nuclei, mvlm medial projection of the ventral longitudinal muscle, pcm pharyngeal circular muscle, pddm pharyngeal dorsal diagonal muscle, pdm posterior diagonal muscle, pdlm pharyngeal dorsal longitudinal muscle, ph pharynx, pps posterior pharyngeal sphincter, pt primary tube, rpm radial pharyngeal muscles, scm semi-circular muscle, st secondary tube, tdm tube diagonal muscle, vlm ventral longitudinal muscle, vllm ventro-lateral longitudinal muscle, vlm ventral longitudinal muscle along the primary tube. The dorsal longitudinal muscle posterior sphincter is more prominent with 8 μm thick- supplies the anterior third of the secondary tube. ness and a diameter of 23 μm. Supplementary circular muscles of the adhesive glands Diagonal muscles (cmag, Figs. 2h, i, k and 3a, b, g, h) are present in the The head diagonal muscle (hdm, Figs. 2j, p and 3a, b, c) tubes, forming a muscular layer around the large adhesive forms a V-shape with two medially joined branches. The glands (ag, Fig. 2k). They thereby supply two cavities, one median part of the muscle is situated in the midline of for each tube. The muscular layer surrounding the the body in the posterior region of the head while the primary tube is smaller than the one surrounding the two extremities extend to the antero-lateral region of secondary tube, and both structures are connected. This the head. suggests that both tubes are supplied by a single set of At the dorso-posterior pharynx, a pair of pharyngeal glands controlled by muscles. Three adjacent nuclei are dorsal diagonal muscles decussate (pddm, Figs. 2p and found within the layer of circular muscles at the base of 3b, d), ventral to the pharyngeal dorsal longitudinal the gland (agn, Fig. 2k), near the anus, around U88. muscle and the dorsal longitudinal muscle. Although their orientation is similar to that of the helicoidal mus- Semi-circular muscles cles, their exclusively dorsal extension and their greater Ventrally opened semicircular muscles (scm, Figs. 2l and width differ significantly from those of the helicoidal 3a,b,f,g)arepresentintheposteriorpartofthespecimen, muscles. but do not extend into the tubes. They originate ventrally, Three diagonal muscles are found in the furca, ex- from each side of the body, and extend to the dorsal side. tending from one side to the contralateral one. The From there, they project to the contralateral side, external twoanteriormuscles(tmd,Figs.2a,b,h,i,kand3a, to the longitudinal musculature. They are more numerous b, g, h) extend from the midline at U86, halfway to in the posterior region, anterior to the furca, where they are the primary and the secondary tube, respectively. The separated by 2–5 μm. Semicircular muscles are less numer- posterior diagonal muscle (pdm, Figs. 2i, k, and 3a, b, ous and spaced further apart (5–8 μm) in the anterior re- h) extends from U89 laterally into the first third of gion of the ovary. They appear to supply only the ovary thesecondarytube. region; their contraction probably reduces the body diam- eter and may be involved in the movement/release of eggs. Circular muscles Pharyngeal circular muscles (pcm 2E,M,O,P and 3A,B,C,D) Dorso-ventral muscles are present around the pharynx. These muscles are numer- Numerous thin muscles (1–2 μm in diameter) traverse the ous(ca.110inonespecimen),positionedproximaltoeach entire trunk dorso-ventrally (dvm, Figs. 2a–c, e-h, k, l, n other, and 1–1.50 μm thick with increasing diameter to- and 3a, b, e-g). These dorso-ventral muscles are spaced wards the posterior region. approximately 5 μm apart in the region between U18 and Two sphincters are present at each extremity of the U95. In this region, two pairs are found laterally in trans- pharynx: one anterior pharyngeal sphincter (aps, Figs. 2b, verse sections of the pharyngeal region (one external and o, p and 3a, b) located just posterior to the mouth, and one more internal pair, dvm, Figs. 2d, e and 3a, b). This one posterior pharyngeal sphincter (pps, Figs. 2m, o, p number increases more posteriorly in the trunk, where up and 3a, b) marking the transition between the intestine to five pairs of dorso-ventral muscles can be detected and the pharynx. The anterior sphincter is smaller, being (dvm, Figs. 2g, k, l, and 3a, b, f). The dorso-ventral 1 μm thick and has a diameter of 17 μm, while the muscles extend dorso-ventrally between the different Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 10 of 29

Fig. 3 (See legend on next page.) Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 11 of 29

(See figure on previous page.) Fig. 3 Schematic drawings of the musculature of Diuronotus aspetos. Anterior end is pointing at the top for a and b, dorsal side is pointing at the top for c–h. a Ventral view of the musculature, b dorsal view of the musculature, c–h cross section of the specimen, c in the head, d posterior part of the pharynx, e anterior of the trunk, f posterior of the trunk, g post-anal region of the trunk, h and in the furca before bifurcation of the tubes. Note that in c and d, the helicoidal pharyngeal musculature is represented in dash lines due to the uncertainty of its presence, and it is not drawn in (a and b). aps anterior pharyngeal sphincter, cmag circular muscle of the adhesive gland, dlm dorsal longitudinal muscle, dvlm dorsal projection of the ventral longitudinal muscle, dvm dorso-ventral muscle, hdm head diagonal muscle, hm helicoidal muscle, int intestine, lplm lateral pharyngeal longitudinal muscle, lvlm Lateral extension of the ventral longitudinal muscle, mvlm medial projection of the ventral longitudinal muscle, ov ovary, pcm pharyngeal circular muscle, pdm posterior diagonal muscle, pddm pharyngeal dorsal diagonal muscle, pdlm pharyngeal dorsal longitudinal muscle; pps posterior pharyngeal sphincter, rpm radial pharyngeal muscles, scm semi-circular muscle, tdm tube diagonal muscle, vllm ventro lateral longitudinal muscle, vlm ventral longitudinal muscle longitudinal muscles and the ciliary bands in various com- Stomatogastric nervous system The stomatogastric binations. However, they are never found external to the nervous system, confined to the pharynx, consists ventro-lateral longitudinal muscles or between the pair of mainly of three main longitudinal nerves: a dorsal (dpn, dorsal longitudinal muscles. Figs. 4a, d–h, j–n and 5a, b, e) and two ventro-lateral nerves (vpn, Figs. 4b, f–h, l–n and 5e), extending basally Nervous system along the midline of each row of radial muscles (Fig. 4). The nervous system of Diuronotus aspetos is described The nerves are closely related to three structures: kino- from acetylated α-tubulin-like immunoreactivity (LIR, cilia, anterior pharyngeal glands and a pharyngeal canal Figs. 4, 5 and 6), serotonin-LIR (Fig. 7) and FMRF- system. The dorsal pharyngeal nerve (dpn, Figs. 4a, d–h, amide-LIR (Fig. 8) (all different LIR of the head region k–o and 5a, b, e) originates at the mouth, where it sup- are summarized in Fig. 9). Similar to previously investi- plies a buccal nerve ring (bnr, Figs. 4a, b and 5a), encirc- gated Gastrotricha, the nervous system consists of paired ling the mouth (probably innervating the anterior nerve cords, which originate from a bilobed dorsal brain, sphincter (aps, Figs. 2b, o, p and 3a, b) opening and clos- and extend posteriorly. In the following section, previously ing the mouth). At U4, two anterior diagonal pharyngeal described and undescribed structures are detailed, such as: nerves (adpn Figs. 4a and 5a) originate from the dorsal i) multiple pairs of longitudinal nerve projections in the nerve, extend antero-laterally to the anterior edges of head (danp, dlpn, hln, Figs. 6a, b, e, j and 9a, b); ii) paired the pharynx and medially join back the dorsal nerve. At anterior ventro-median nerves (avmn, Fig. 6a, d and 9b); iii) U3, one pharyngeal dorso-ventral nerve (pdvn, Figs. 4a, dorsal nerves posterior to the brain (hdpn, Figs. 6a, h and b and 5a) originates from each of the anterior diagonal 9a); iv) paired ventro-lateral nerve cords (vlnc, Figs. 6b-d, f, nerve, and supply ventrally a pharyngeal gland longitu- g, k and 9b); v) paired posterior nerves, projecting into the dinal nerve (plgn, Figs. 4b and 5c) innervating an anter- adhesive tubes (nppt, Fig. 6k), vi) bilobed, dorsal brain with ior pharyngeal gland (apg, Figs. 4a, c, i and 5c) (which three commissures (main neuropil and anterior ventral and opens into the mouth). On the right side of the speci- dorsal commissure, together forming a nerve ring encirc- men, a dorso-anterior pharyngeal canal nerve (dpcn, ling the pharynx) (np and anr, Figs. 6a-c, e, h-j and 9a, b); Figs. 4a, d, j and 5a) extends posteriorly from the anter- vii) two pairs of ganglia along the nerve cord: one anterior ior diagonal nerve, possibly supplying the asymmetric and one terminal (pgg and ang, Figs. 6b, f, k and 9b); viii) dorsal pharyngeal canal (dpc, Figs. 4a, f, g, l, m and 5e). four ventral trunk commissures (spc, tvc, pac and pco, At U9 a pharyngeal nerve ring (pnr, Figs. 4a, b, e, k and Figs. 6b, d, g, k and 9b); and ix) a pharyngeal nervous sys- 5b, d) supplying the ventral and dorsal nerves is present. tem, consisting of three longitudinal nerves (one per A pair of paramedian dorsal pharyngeal nerves (dpnp, pharyngeal row of radial muscles) and supplementary Figs. 4a, f, l and 5e) originates from the dorsal nerve at minor nerves (Figs. 4 and 5). Additionally, the serotonin- U19 and extends in a parallel fashion on each side, to LIR and FMRF-amide-LIR gave very detailed results, fuse again with the dorsal nerve at U26 (Figs. 4a, g, m allowing us to collect precise data on the number, position and 5e). The dorsal nerve extends more posteriorly and connection of perikarya (Figs. 7, 8 and 9). where it innervates a two-celled pharyngeal posterior cluster (ppc, Figs. 4a and 5i) at the posterior margin of Acetylated α-tubulin-like immunoreactivity (acetylated the pharynx. Two nerves extend the terminal part of α-tubulin-LIR) the ventro-lateral pharyngeal sections: the lateral Acetylated α-tubulin-LIR provides information on most gland longitudinal nerve (plgn, Figs. 4a and 5c), and a neurites, cilia as well as other portions of cytoskeletons median kinocilium longitudinal nerve (plkn, Figs. 4b, of the cells. However, not all minor neurites of the ner- c and 5g), with the latter supplying a mouth and a vous system are traced and the description focuses on pharyngeal kinocilia, respectively, at U1 and U6 (mk the central nervous system and sensory structures. andpk,Figs.4b-d,j,iand5c,g).Theglandand Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 12 of 29

Fig. 4 Pharyngeal nervous system and canal system of Diuronotus aspetos. a, b Anterior is pointing at the top; c–n dorsal is pointing at the top. a–h Schematic drawings with nerves in blue and pharyngeal system in yellow, nuclei in grey, glands in green and cilia in red. a Dorsal section of the pharynx. b Ventral section of the pharynx. c–h Successive transverse sections of the pharynx from anterior to posterior. i–n CLSM virtual transverse sections at the same levels as (c–h). Acetylated α-tubulin-LIR in glow and DAPI in cyan. adpn, anterior diagonal pharyngeal nerve, apg anterior pharyngeal gland, avrc anterior ventro-median right pharyngeal canal, bnr buccal nerve ring, dpc dorsal pharyngeal canal, dpcn dorso- anterior pharyngeal canal nerve, dpn dorsal pharyngeal nerve, lpvc left posterior ventro-median canal, mk mouth kinocilium, pdvn pharyngeal dorso-ventral nerve, pk posterior pharyngeal kinocilium, plgn pharyngeal longitudinal gland nerve, plkn pharyngeal longitudinal kinocilium nerve, pmdn paramedian dorsal pharyngeal nerves, pnr pharyngeal nerve ring, ppc posterior pharyngeal cluster, rpvc right posterior ventro-median canal, vlpc ventro-lateral pharyngeal canal, vlpg ventro-lateral pharyngeal ganglion, vpn ventral pharyngeal nerve Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 13 of 29

Fig. 5 (See legend on next page.) Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 14 of 29

(See figure on previous page.) Fig. 5 CLSM of the pharyngeal nervous system and canal system of Diuronotus aspetos. a–c, g, i anterior is pointing at the top. d–f, h anterior pointing left. CLSM maximum intensity projection of sub-stacks. Acetylated α-tubulin-LIR in glow, DAPI in cyan. a Dorso-anterior section of the pharynx. b Dorso-anterior section of the pharynx, more ventral than b). c Ventro-anterior section of the pharynx. d Ventro-posterior section of the pharynx. e Dorso-posterior section of the pharynx. f Medio-posterior portion of the pharynx. g Medio-anterior section of the pharynx. h Details of the ventro- lateral pharyngeal ganglion. i Details of the posterior pharyngeal ganglion. adpn anterior diagonal pharyngeal nerve, anr anterior nerve ring, apg anterior pharyngeal gland, avrc anterior ventro-median right pharyngeal canal, bnr buccal nerve ring, dpc dorsal pharyngeal canal, dpcn dorso-anterior pharyngeal canal nerve, dpn dorsal pharyngeal nerve, hdpn head dorso-posterior nerve, lpvc left posterior ventro-median canal, mk mouth kinocilium, np neuropile, pdvn pharyngeal dorso-ventral nerve, pk posterior pharyngeal kinocilium, plgn pharyngeal longitudinal gland nerve, plkn pharyngeal longitudinal kinocilium nerve, pmdn paramedian dorsal pharyngeal nerves, pnr pharyngeal nerve ring, ppc posterior pharyngeal cluster, rpvc right posterior ventro-median canal, ss sensoria, vlpg ventro-lateral pharyngeal ganglion, vlpc ventro-lateral pharyngeal canal, vpn ventral pharyngeal nerve kinocilium nerves originate at U7 from an elongated (LI-reactive) and posterior serotonin-LI-reactive) nerves, ventro-lateral pharyngeal ganglion (vlpg, Figs. 4 and 5h, f), which eventually fuse dorso-laterally, forming the lateral consisting of three nuclei and extending from U7 to U12 sections of the anterior nerve ring. One anterior and one (probably integrating the signal collected by the two kino- posterior short longitudinal nerves (cpn, Figs. 6b, i and ciliaandresponsiblefortheputativeterminalglandsecrete 9b) extend from the ventral portion of the anterior nerve release). Moreover, the ganglion seems to be related to the ring, innervating two median ciliary patches (described ventro-lateral pharyngeal canal (vlpc, Figs. 4a, b, e-g, k-n below). The neuropil supplies ventrally a pair of anterior and 5f, i) described below. The ventral pharyngeal nerves ventro-median nerves (avmn, Figs. 6b, d, and 9b) ex- (vpn, Figs. 4b, f, g, l, m and 5d) (supplied by perikarya at tending between the anterior nerve ring and the post- U15 and U19) extend from the ganglion, until U28. pharyngeal ganglion posteriorly (pgg, Figs. 6b, f and 9b). Due to the unknown nature of the canal system and its It extends parallel to the pharyngeal median ciliated cell main acetylated α-tubulin-LIR (as well as a weak FMRF- (pmcc, Fig. 10b, g), probably innervating it. Two pairs of amide-LIR), it is described in this nervous system section. dorso-median anterior nerves projections (danp, Figs. 6a, It consists of radially flattened cavities, sometimes asym- j and 9a) and two pairs of dorso-lateral anterior nerve metrical, extending longitudinally within the pharynx projections (dlnp, Figs. 6a, j and 9a) extend from the an- (Figs. 4a, b, e, f, k, l and 5d, e). Six pharyngeal canals terior nerve ring and the lateral sides of the neuropil, re- extend the pharynx: i) the unpaired right ventro-anterior spectively, projecting anteriorly. One pair of head lateral pharyngeal canal (avrc, Figs. 4b, e, f, k, l and 5d) extending nerves (hln, Figs. 6a, b, e, j and 9a, b) extends from the from U6 to U26; ii–iii) The paired ventro-lateral lateral sides of the neuropil and bifurcates, posteriorly pharyngeal canals (vlpc, Figs. 4a, b, e-h, k-n and 5f, i), supplying a cell with a large and diffuse nucleus (pos- extending from U7 (at the level of the pharyngeal ganglia sibly a gland cell (lgcb, Figs. 6a, e and 9a)), and anteri- (vlpg, Figs. 4a and 5h, f)) to U30, and merging dorso- orly forming a nerve projection. Each of these anterior posteriorly; iv-v) the paired ventro-posterior pharyngeal nerve projections probably innervates head sensory or- canals extending from U24 for the left one (lpvc, Figs. 4b, gans. Dorso-laterally, the posterior sides of the neuropil g, h, m, n and 5d) to U28 and from U26 for the right one supply the ventro-lateral nerve cord (vlnc, Figs. 6b, c, d, (rpvc, Figs. 4b, g, h, m, n and 5d) to U28; vi) the dorsal f, g, j, k and 9b) of D. aspetos, which extends along the pharyngeal canal (dpc, Figs. 4a, f, g, l, m and 5e) extending entire length of the specimen adjacent to the lateral lon- along the right side from U6 to U15, then reaching the gitudinal ciliary bands and the ventro-lateral longitudinal midline and bifurcating in two symmetrical branches, muscle, probably innervating these two structures. Two following the paramedian dorsal nerves of the phar- head dorso-posterior nerve (hdpn, Figs. 5e, 6a, h and ynx between U19 and U27. Few nuclei are embedded 9a), extending along the pharynx, eventually supply the in the pharyngeal canal system (Fig. 4a, b, g). post-pharyngeal ganglion. They may innervate the anter- ior portion of the dorsal longitudinal muscle. A pair of Central nervous system The neuropil (np, Figs. 6a, c, e, head diagonal nerves (hdn, Figs. 6a, h and 9a) originates h, j and 9a) is 14 μm thick and its center is positioned at dorso-laterally of the neuropil, decussate dorsal to the U15. One 3 μm broad nerve extends antero-medially pharynx at U22, and each extend ventro-laterally to a from the neuropil and branches laterally to form a dorsal single perikaryon at U23. Comparison across specimens and a ventral commissure at U12 and U9, respectively, suggests that the position of these diagonal nerves corre- together constituting an anterior nerve ring (anr, Figs. 5d, sponds to the position of the pharyngeal dorsal diagonal 6a-c, i, j and 9a, b). At the dorsal section of the anterior muscle (pddm, Figs. 2p and 3b, d), which it probably in- nerve ring (anr, Fig. 9), the acetylated α-tubulin-LIR is nervates. At U27 and U29, two thin nerves originate relatively weak, and the commissure consists of two from the anterior ventro-median nerve, and form to- transverse (anterior FMRF-amide-like-immunoreactive gether at U28 a sub-pharyngeal commissure (spc, Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 15 of 29

Fig. 6 (See legend on next page.) Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 16 of 29

(See figure on previous page.) Fig. 6 Drawing and CLSM of the acetylated α-tubulin-LIR nervous system of Diuronotus aspetos. Anterior pointing left for a–i, and pointing at the top for j and k. a, b Schematic drawings of the α-tubulin-LIR of the anterior part of the specimen: nerves in blue, nuclei in grey, and opposite ventral or dorsal nervous system in light grey a dorsal b ventral. c CLSM ventral view of the maximum intensity projection (MIP) of the entire specimen. d–k CLSM MIP sub-stacks of various parts of the specimen. Acetylated α-tubulin-LIR in glow, and DAPI in cyan in all CLSM pictures. d Ventro-anterior nervous system. e Neuropil side f Ventral, post pharyngeal ganglion. g Ventral, trunk commissure. h Dorso-posterior part of the head i ventro-anterior part of the head j Dorso-anterior part of the head. k Ventro posterior terminal part of the specimen. ang anal ganglion, anr anterior nerve ring, avmn anterior ventro-median nerve, br brain, cpn ciliated patch nerves, danp dorso-median anterior nerve projection, dlnp dorso-lateral anterior nerve projections, hdpn head dorso-posterior nerve, hdn head diagonal nerve, hln head lateral nerve, lgcb lateral gland cell of the brain, np neuropile, nppt nerve projection of the primary tube, pac pre-anal commissure, pco posterior commissure, pgg post-pharyngeal ganglion, ph pharynx, spc sub-pharyngeal commissure, tt, testis, tvc trunk ventral commissure, vlnc ventro-lateral nerve cord

Figs. 6b, d and 9b). At U50, anterior to the testis, a thin serotonergic-LI-reactive neurites, extends ventrally to trunk ventral commissure (tvc, Fig. 6g) is present. At U84, supply a serotonin-LI-reactive para-pharyngeal cluster an anal ganglion (ang, Fig. 6k) of 6–8 cells supplies a pre- (sppc, Figs. 7b-e and 9b) with three perikarya, and anal commissure (pac, Fig. 6k). Posterior to the anus, at extends to the posterior end forming the posterior U87, the two ventro-lateral nerve cords form the posterior commissure. Additionally, single serotonin-LI-reactive commissure (pco, Fig. 6c, k), from which two nerve perikarya of the post-pharyngeal ganglion and of the projections of the primary tube (nppt, Fig. 6c, k) extend. anal ganglion are present respectively at U33 and U93 (spog, and spag, Fig. 7b, c, f) as well as nerve projections Serotonin-like immunoreactivity (serotonin-LIR) of the primary tube (snpt, Fig. 7c, f). The nervous system shown by serotonin-LIR consists of a dorsal neuropil, the anterior nerve ring, anterior and FMRF-amide-like immunoreactivity (FMRF-amide-LIR) posterior projections, the two ventro-lateral nerve cords, The FMRF-amide-LI-reactive nervous system consists of one posterior commissure as well as several perikarya. the brain neuropil, the anterior nerve ring, the anterior Three main commissures (an anterior (sacn), a median ventro-median nerve, the ventro-lateral nerve cord, the (smcn) and a posterior (spcn)) in the brain neuropil sub-pharyngeal commissure and the posterior commis- show serotonin-LIR (Fig. 7a, d, e) as well as an isolated sure. Different parts of the nervous system show varying patch postero-median to the neuropil (spp, Fig. 7a, d, e). immunoreactivity intensities, as illustrated in Fig. 8. Three longitudinal nerves showing serotonin-LIR are The neuropil (fnp, Fig. 8a, c, d, f, g) consists of four found in the brain: i) the median-most brain nerve connectives: two anterior and two posterior, supplied by (smbn, Fig. 7a, d), ii) the paramedian brain nerve (spbn, several posterior and lateral perikarya. Antero-laterally Fig. 7a, d), and iii) the lateral brain nerve (slbn, Fig. 7a, to the neuropil, one pair of perikarya supplies a very d). Four very thin lateral nerves of the posterior com- short dorso-lateral anterior nerve projections (fpp, missure of the neuropil (slpn, Fig. 7a, d) form complex Fig. 8a, d, f, g) (corresponding to the base of the acety- connections with the other nerves of the brain as well as lated α-tubulin-LI-reactive projections (dlnp; Figs. 6a, j to the dorso-median perikaryon (sdmp, Fig. 7a, c, d, e). and 9a)). Additionally, three lateral perikarya of the brain A postero-lateral nerve node (spln, Fig. 7a, d, e) is (flpb, Fig. 8a, f, g) and a pair of dorso-posterior clusters present postero-laterally to the neuropil, being formed of the brain (fdpc, Figs. 8a, f, g and 9a) with three by the merging of several nerves, and supplies the perikarya, are present. Comparisons between differently ventro-lateral nerve cord (slnc, Fig. 7b, c, f). One dorso- stained specimens, and use of DAPI, enabled us to infer lateral perikaryon (sdlp, Fig. 7a, c, d, e) supplies the that the postero-median cell of the FMRF-amide-LI-react- postero-lateral nerve node, with a short nerve. The ive dorso-posterior cluster corresponds to the serotonin- median-most brain nerve extends anteriorly from the LI-reactive dorso-posterior perikarya (sdmp, Fig.7; fdpc, posterior of the neuropil until U6 (Fig. 7a). The lateral Figs. 8a and 9a). Anteriorly, the neuropil supplies the an- brain nerve is short and extends from the postero-lateral terior ventro-median nerve (fvmn, Figs. 8b, i and 9b), also nerve node, being supplied by some of the commissures supplied by two ventro-lateral perikarya of the brain (fvpb, of the neuropil (Fig. 7a). The paramedian brain nerve Fig. 8a, e, i) and a ventral perikaryon of the anterior nerve originates from the postero-lateral nerve node and sup- ring (fvpr, Fig. 8b, i). The nerve ring (fanr, Fig. 8a-d, g, h, j) plies the serotonin-like-LI-reactive anterior nerve ring is supplied by one anterior and one posterior unpaired (sanr, Fig. 7a-d), subsequently extending more poster- dorso-median perikarya (fpar, Fig. 8a, d, f, h). Ventro- iorly as an anterior nerve projection until U4 (Fig. 7a, d). posterior to the neuropil, a pair of tricellular clusters also The anterior nerve ring consists dorsally of two and ven- supply the ventro-median nerve (fvnc, Fig. 8n, i), which trally of one serotonin-LI-reactive nerves (sanr, Fig. 7a, extends further posterior until the two FMRF-amide-LI- b). The ventro-lateral nerve cord, consisting of two reactive perikarya of the post-pharyngeal ganglion (fppg, Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 17 of 29

Fig. 7 serotonin-LIR nervous system of Diuronotus aspetos. The anterior is pointing left for all figures. a, b Schematic drawings of the serotonin-LIR of the anterior part of the specimen: nerves and perikarya in green, nuclei in grey, and opposite ventral or dorsal nervous system in light grey. a Dorsal view, b ventral view. c–f CLSM images with serotonin-LIR in glow. c CLSM maximum intensity projection (MIP) of the entire specimen. d Dorsal MIP of the brain e CLSM sub-stack MIP showing details of the brain perikarya f CLSM sub-stack MIP of the ventro-posterior terminal part of the specimen. br brain, ph pharynx, sacn serotonin-LI-reactive anterior commissure of the neuropil, sanr serotonin-LI-reactive anterior nerve ring, sdlp serotonin-LI-reactive dorso-lateral perikaryon, sdmp serotonin-LI-reactive dorso-median perikaryon, slbn serotonin-LI-reactive lateral brain nerve, slnc serotonin-LI-reactive ventro-lateral nerve cord, slpn serotonin-LI-reactive lateral nerves of the posterior commissure of the neuropil, spln serotonin-LI-reactive postero-lateral nerve node, smbn serotonin-LI-reactive median-most brain nerve, smcn serotonin-LI-reactive median commissure of the neuropil, snp serotonin-LI-reactive neuropil, snpt serotonin-LI-reactive nerve projection of the primary tube, spag serotonin-LI-reactive perikarya of the anal ganglion, spbn serotonin-LI-reactive paramedian brain nerve, spcn serotonin-LI-reactive posterior commissure of the neuropil, spco serotonin-LI-reactive posterior commissure, spog serotonin-LI-reactive perikarya of the post-pharyngeal ganglion, spp serotonin-LI-reactive neuropil patch, sppg serotonin-LI-reactive para-pharyngeal cluster Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 18 of 29

Fig. 8 (See legend on next page.) Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 19 of 29

(See figure on previous page.) Fig. 8 FMRF-amide-LIR nervous system of Diuronotus aspetos. Anterior is pointing left for a–g and i–l and dorsal pointing at the top for h. a, b Schematic drawings of the FMRF-amide-LIR of the anterior part of the specimen: nerves in magenta, nuclei in grey, and opposite ventral or dorsal nervous system in light grey. a Dorsal view, b ventral view. c CLSM dorsal view of the maximum intensity projection (MIP) of the entire specimen. d–l (Except h) CLSM sub-stack MIP of various parts of the specimen. FMRF-amide-LIR in glow, and DAPI in cyan in all CLSM pictures. d Dorsal view of the whole neuropil. e Ventral part of the brain. f and g different levels of the dorsal part of the neuropil. h CLSM virtual transverse section of the anterior nerve ring. i Ventro-anterior part of the head. j Ventral commissure of the anterior nerve ring. k Ventral post-pharyngeal ganglia. l ventro-posterior terminal part of the specimen. Anterior of the specimen on the left for a-g and i-l and dorsal on top for h. br brain, egg egg, fanr FMRF-amide-LI-reactive anterior nerve ring, fapn FMRF-amide-LI-reactive anterior perikarya of the ventro-lateral nerve cord, fdpc FMRF-amide-LI-re- active dorso-posterior cluster of the brain, flnc FMRF-amide-LI-reactive ventro-lateral nerve cord, flpb FMRF-amide-LI-reactive lateral perikarya of the brain, fnp FMRF-amide-LI-reactive neuropil, fnpt FMRF-amide-LI-reactive nerve projection of the primary tube, fpar FMRF-amide-LI-reactive dorso-median perikarya of the anterior nerve ring, fpco FMRF-amide-LI-reactive posterior commissure, fpp FMRF-amide-LI-reactive perikarya of the dorso-lateral anterior nerve projections, fppg FMRF-amide-LI-reactive post-pharyngeal ganglion, fspc FMRF-amide-LI-reactive sub-pharyngeal commissure, fvmn FMRF-amide-LI-reactive anterior ventro-median nerve, fvnc FMRF-amide-LI-reactive anterior ventro-median nerve cluster, fvpb FMRF-amide-LI-reactive ventro-lateral perikarya of the brain, fvpr FMRF-amide-LI-reactive ventral perikarya of the anterior nerve ring, ph pharynx

Fig. 8b, c, k). The paired ventro-lateral nerve cords (flnc, lateral row of cells is present, which extends until the Fig. 8b, c, i, k, l) is supplied by the postero-lateral part of posterior trunk, as described originally [17]. the neuropil and by three anterior perikarya (fapn, Fig. 8b, The two ventro-median ciliary patches at U7 and U11 c, e, i) (two anterior and one posterior, separated by (acp, pcp, Fig. 10b, h, i, position of patches measured from 8 μm). The FMRF-amide-LI-reactive sub-pharyngeal com- the center) are innervated by two short diffuse 5 μmwide missure (fspc, Fig. 8b, i), ventro-lateral nerve cord, poster- longitudinal nerves (cpn, Figs. 6b, i and 7b), joining per- ior commissure (fpco, Fig. 8c, l), and nerve projections of pendicularly the anterior nerve ring. Each patch also shows the primary tube (fnpt, Fig. 8c, l) follow the description of an acetylated α-tubulin-LI-reactive positive ring around the the acetylated α-tubulin-LIR. ciliated area. The divergent morphology of these anucleate multiciliated cells and their close relation to the nervous Ciliation system suggest that they could be sensory structures. The locomotor ciliation consists of a dense ventro-anterior Several sensoria are scattered along the body (ss, ciliated area and two thin ciliated bands, which are extend- Fig. 10d, j), and two pairs of pharyngeal sensory cilia ing to the posterior part of the specimen at U87 (Fig. 10c). (mk and pk, Figs. 3a-d, i, j, 4c, g and 10i) are located in This general pattern supports the original description of the pharyngeal region (see the nervous system section Todaro et al. [17], although numerous details can be added. for further details). CLSM allowed the identification of individual multiciliated Two pairs of nephridia are found along the body cells and determination of their precise pattern. (Fig. 10d): the anterior pair is situated ventro-laterally and Dorsally, the muzzle is covered by two transverse rows the posterior pair is located dorso-laterally, relatively close of multiciliated cells. The anterior row consists of three to the midline. The anterior pair of protonephridia (apn, pairs of relatively small head dorso-anterior ciliated cells Fig. 10d, j) is situated anterior to the testis at U42 and the (3 μm, hacc, Fig. 10a, e, f), while the posterior row is cilia are 20 μm long. The posterior pair of protonephridia constituted by a pair of larger head postero-lateral dorsal (ppn, Fig. 10d, k) is situated at U74 with 15 μm long cilia. ciliated cells (hpcc, Fig. CA,E) and a head dorso-median Each nephridium seems to possess two straight coaxial ciliated cell (hmcc, Fig. CA,E) of similar size (6 μm). The cilia (c, c,’ Fig. 10j, k), thus resembling the general paucitu- pattern of the head lateral ciliated cells (hlcc, Fig. 10a, f) bulatinian protonephridia with its two adjacent monoci- could not be resolved in details due to the dorso-ventral liated terminal cells projecting into a non-ciliated canal mounting of the specimen. However, at least four cells cell and ending with a nephridiopore epidermal cell [2, at the dorso-lateral level are present, and probably the 41]. The canal cell and the nephridiopore cell have not same number at the ventro-lateral level. been stained, which is why information on the orientation The ventral head bears 20 multiciliated cells organized and opening of protonephridia in D. aspetos is lacking. in four paired longitudinal rows and one median row, containing 2,2,3,2,2,2,3,2,2 cells (Fig. 10b). Posterior to Discussion the head, ventral to the pharynx, from U9, only two ad- Phylogeny jacent rows of multiciliated cells are present on each The present phylogenetic analysis confirms that Diuro- side, containing one large pharyngeal median ciliated cell notus aspetos belongs to the monophyletic family Musel- (pmcc, Fig. 10b, g, 45 μm long) and three pharyngeal lat- liferidae as proposed previously based on morphology eral ciliated cells (plcc, Fig. 10b, g, h, 20–35 μm long), [22, 23, 26]. We furthermore find Xenotrichulidae sister respectively. Posterior to the pharynx, only one paired group to Muselliferidae (100 % support), opposed to its Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 20 of 29

Fig. 9 (See legend on next page.) Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 21 of 29

(See figure on previous page.) Fig. 9 Schematic drawing of acetylated α-tubulin-LIR, FMRF-amide-LIR and serotonin-LIR nervous system of Diuronotus aspetos, showing the correspondences between the different nervous systems. Anterior pointing at the top. Acetylated α-tubulin-LIR nervous system in blue, FMRF- amide-LIR nervous in magenta, and serotonin-LIR nervous system in green. Cell nuclei in grey and opposite nervous system in light grey. Legends in bold indicate structures showing-LIR for at least two molecules tested. a Dorsal, and b ventral. anr anterior nerve ring, avmn anterior ventro median nerve cord, br brain, cpn ciliated patch nerves, danp dorso-median anterior nerve projection, dlnp dorso-lateral anterior nerve projections, fdpc FMRF-amide-LI-reactive postero-lateral brain cluster, fvnc FMRF-amide-LI-reactive ventro-median nerve cluster, hdn head diagonal nerve, hln head lateral nerve, hdpn head dorso-posterior nerve, hpdn head diagonal nerve, np neuropile, pgg post-pharyngeal ganglion, ph,pharynx,spc sub- pharyngeal commissure, sppc serotonin-LI-reactive para-pharyngeal cluster, vlnc ventro-lateral nerve cord position next to Group B (“Chaetonotidae” + Dasytydae in D. aspetos. The ventro-lateral longitudinal muscle of D. + Neogosseidae) in Kånneby et al. [26] (69 % PP). More- aspetos can be homologized with the somatic ventro-lateral over, the placement of D. aspetos considerably reduces muscle (or musculus lateralis) of other Gastrotricha, and the internal branch length of Muselliferidae, diminishing the ventral longitudinal muscle resembles those found in the possibility of long-branch attraction, with e.g. Neo- the paucitubulatinian Musellifer delamerei, Xenotrichula dasys ([49] and present study) or Dactylopodola [26], intermedia Remane [57] and Heteroxenotrichula squamosa and the support of group B is now maximum. We fur- Wilke, 1954 [22, 58] (Fig. 11). ther note two other interesting points: the sister group The unique semi-circular muscles of D. aspetos may relationship between Neogosseidae and Dasydytidae is aid the oviposition together with the dorso-ventral mus- recovered [50], and the sister group relationship of mar- cles, hereby functionally replacing the dorso-dermal ine Aspidiophorus to the remaining members of the longitudinal muscle split enveloping the egg in other Group B is found again, similarly to Kånneby et al. 2012 Paucitubulatina [22]. They likely act as the posterior [49], but not Kånneby and Todaro [50]. complete circular muscles found in the region of the sex- ual organs of Neodasys cf. uchidai Remane, 1961 [59, 60]. Musculature Functionally similar circular muscles are also found in the The overall musculature of Diuronotus aspetos is relatively meiofauna gnathiferan Gnathostomula armata Riedl [61] simple, consisting of only three pairs of longitudinal mus- and Gnathostomula peregrina Kristeuer [62] (Gnathosto- cles in the trunk as well as a unique arrangement of mul- mulida), here arranged in dense pattern around the tiple dorso-ventral muscles. The number of pairs of posterior male organs [63, 64]. longitudinal muscles in D. aspetos is inferior to what is The evolution of the dorso-ventral muscles of Chaeto- found in most Paucitubulatina, having at least five, often notida as deriving from the circular musculature has been six, pairs of longitudinal muscles, that are often distrib- one of the central debates in previous studies [22, 31]. In uted as three pairs of splanchnic and three pairs of Macrodasyida and Multitubulatina, the circular muscula- somatic muscles (Musellifer, Draculiciteria, Heteroxenotri- ture consists of splanchnic and somatic elements, the chula, Xenotrichula, Chaetonotus, Aspidiophorus,and former encircling the intestine and the latter, which Polymerurus) [22, 31, 32] (Fig. 11). The previously pro- derives from splanchnic elements, encircles the ventro- posed hypothetical ancestral state of musculature in lateral longitudinal muscle on both sides [2, 22, 59]. In Paucitubulatina [2] shows a split of the dorsal longitudinal Paucitubulatina, the trunk circular muscles are either ab- muscle (musculus dorsalis) into two branches, not present sent or have a dorso-ventral orientation (i.e. incomplete in D. aspetos that instead has a branch of the ventral lon- circular muscles). Such dorso-ventral muscles are found gitudinal muscle running dorsally. The more complex in Xenotrichulidae and Muselliferidae (group A, Fig. 1) [2, branching pattern of the ventral longitudinal muscle may 22, 31, 65] (Fig. 11). Hence, if the group A is monophy- be an adaptation to the large size of D. aspetos, compen- letic, it means that this arrangement is a synapomorphy of sating for the low number of longitudinal muscle. this group, and that the group B lost the dorso-ventral, or The helicoidal musculature encircles the dorsal longitu- circular, muscles (regained in the genus Polymerurus [32]). dinal muscles but not the ventral longitudinal muscles or Comparatively, D. aspetos is the only Paucitubulatina with the ventro-lateral longitudinal muscles. The relative pos- more than two sets of dorso-ventral muscles in the trans- ition of the dorsal longitudinal muscle indicates a homology verse axis. The median-most dorso-ventral muscles may tothedorsalsplanchnicmuscleofotherPaucitubulatina be homologous with the visceral circular muscles in other (Fig. 11) (see Kieneke and Schmidt-Rhaesa [2] for further gastrotrichs. The lateral sets of dorso-ventral muscles discussion and limitations of this notion). However, its present varying positions relative to the longitudinal more dorsal position indicates that it supports the body muscles, making homologies difficult to assess. Further- wall rather than the digestive tract, perhaps furthermore more, no dorso-ventral muscles are present lateral to the compensating for the missing dorso-dermal muscle branch ventro-lateral longitudinal muscle, which would be an Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 22 of 29

Fig. 10 (See legend on next page.) Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 23 of 29

(See figure on previous page.) Fig. 10 Ciliation of Diuronotus aspetos. Anterior pointing at the top for all figures. a and b drawings of the locomotory ciliation: a dorsal view, b ventral view. c–k CLSM maximum intensity projection (MIP) sub-stacks of the acetylated α-tubulin-LIR. c Ventral view of the whole specimen showing the organization of the locomotor ciliation. d Dorsal view of the whole specimen showing parts of the locomotor ciliation and the position of the protonephridia. e And f, dorsal head ciliation. e Is more dorsal than f. g–i Ventral head and pharyngeal ciliation: g is more dorsal than h which is more dorsal than i. j and k details of, respectively, the anterior and the posterior pairs of protonephridia. acp anterior ciliated patch, apn anterior proto-nephridia, br brain, c, c’ cilia of the proto-nephridia, hacc head dorso-anterior ciliated cells, hlc head lateral ciliation, hlcc head lateral ciliated cells, hmcc head dorso-median ciliated cell, hpcc head postero-lateral dorsal ciliated cell, hvc head ventral ciliation, hvlm head ventral lateral-most row of ciliated cells, hvmm head ventral median-most row of ciliated cells, hvpl head ventral para-lateral row of ciliated cells, hvpm head ventral paramedian row of ciliated cells, mz muzzle, pc pharyngeal ciliation, pcp posterior ciliated patch, ph pharynx, pk posterior pharyngeal kinocilium, plcc pharyngeal lateral ciliated cells, pmcc pharyngeal median ciliated cell, ppn posterior proto-nephridia, ss sensoria, tc trunk ciliation, tcc trunk ciliated cells, tt testis arrangement expected from a derived somatic semi- Stomatogastric nervous system circular muscle such as found in other Paucitubulatina Similar to other Chaetonotida [12, 13, 18], one dorso-me- [20, 22], and see Fig. 11. Consequently, the inner-most dian and two ventro-lateral longitudinal nerves constitute dorso-ventral muscles of D. aspetos can be homologized the overall pharyngeal nervous system of Diuronotus aspe- with the semi-circular muscles of other Paucitubulatina tos. However, the present study finds several additional (Fig. 11), and the arrangement of the other dorso ventral structures previously undescribed for chaetonotids such muscles is so far unique in Gastrotricha [2, 22, 32]. as: i) five additional symmetric and one asymmetric The head diagonal muscle of D. aspetos may be homolo- longitudinal nerves branching off from the main nerves, gous to the head semi-circular muscle found in Musellifer ii) two previously undescribed commissures (anterior- delamerei and Dactylopodola baltica (Remane, 1926) [22, most buccal nerve ring, middle pharyngeal nerve ring), 66, 67] showing the same anterior position and shape and iii) a pair of ventro-lateral pharyngeal ganglia (innerv- though a different orientation. It is however probably only ating anterior sensory structures). homologous to the one found in Musellifer since it is the However, only the pharyngeal nervous system of Cepha- sister group of Diuronotus,whileDactylopodola is a lodasys maximus has been comprehensively described distantly related Macrodasyida (Fig. 1). The posterior [37] and little is known about the pharyngeal nervous diagonal muscle and the diagonal muscle of the tubes re- system of Chaetonotida (but see [12, 13]). Nonetheless, semble a muscle found in the posterior region of Hetero- ultrastructural studies by Teuchert (1877) [73] and xenotrichula squamosa (Fig. 3a, [22]), but no similar Ruppert [18] provide various details of the pharynx in sev- muscle exist in Musellifer delamerei, Xenotrichula inter- eral gastrotrichs, including some details on Diuronotus sp. media or X. punctata Wilke, 1954. The lack of similar In Macrodasyida, the inverted organization of the phar- muscle in the closely related taxa within group A indicates ynx generally offers one ventro-median and two dorso- that this muscle is an apomorphy of Diuronotus (Fig. 11) lateral nerves as well as one additional dorso-median nerve [22, 58]. The so-called cross-over muscles found in [18]. In Turbanella cornuta, an additional asymmetric Macrodasyida with a bilobed caudal end has a similar “thick” ventro-lateral nerve is also present in the pharynx function, being involved in the movement of the posterior [73], which resembles the one short asymmetric dorso- tubes, yet with the lack of presence in other Paucitubula- lateral longitudinal nerve found in D. aspetos to a certain tina and different morphology in D. aspetos it is most degree. Cephalodasys maximus presents a pair of ventro- likely of convergent origin [2, 66, 68]. lateral asymmetric nerves (one short, one long) in the pharynx, but they originate more posteriorly from the Nervous system pharyngeal nerve ring [37]. The probable convergent origin To date, Xenotrichula intermedia and Xenotrichula velox of the asymmetric pharyngeal nerves in the morphologic- Remane [69] are the only other Paucitubulatina for which ally and phylogenetically diverse Macrodasyida C. maximus the nervous system has been studied with CLSM [13], (Cephalodasyidae) and T. cornuta (Turbanellidae) contra- therefore the present study adds valuable information. On dicts their homology with the one found in D. aspetos,but the other hand, the nervous system of Neodasys (Multitu- shows that asymmetry in the pharynx of gastrotrichs might bulatina was described in details with CLSM) [12, 39], as have evolved multiple times in Gastrotricha [15, 29]. well as several Macrodasyida [15, 35, 36, 38, 39, 70, 71]. Macrodasyida and Neodasys possess multiple triplets Furthermore Cephalodasys maximus Remane [67] and of pharyngeal cilia [18], which according to the basal Turbanella cornuta Remane [72] have been described in interrelationship of Gastrotricha suggest that the lack of detail with TEM [37, 73]. This offers a broad, but not reports of these structures in Paucitubulatina represents a comprehensive, literature for comparing the nervous sys- loss (rather than a primary absence). A single short pair tem of D. aspetos with other Gastrotricha. may have either re-appeared in Diuronotus (according the Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 24 of 29

occasionally lined by nerves. However, a single trans- verse TEM micrograph of Diuronotus sp. by Ruppert (Figure 14, [18]), purportedly from the level of the ventro-lateral pharyngeal ganglion, reveals a dorso- lateral as well as three ventro-lateral electron-lucent areas, which most likely resemble the canal system. The system may be unique to Diuronotus or Muselliferidae, and its function is unknown.

Central nervous system The overall morphology of the nervous system of Diuronotus aspetos is similar to other gastrotrichs [2] consisting of a “dumbbell-shaped” dorsal brain with a dorsal neuropil and a pair of ventro-lateral nerves. How- ever, additional nerves and specific perikarya are found in D. aspetos. A summary figure (Fig. 12) shows the evo- lution of the serotonin-LI-reactive nervous system in Chaetonotida, since this is the most comparable immu- nostaining across Gastrotricha.

Longitudinal nerves Anterior to the brain neuropil of Diuronotus aspetos, four pairs of dorsal nerve projec- tions are found (acetylated α-tubulin-LI-reactive, in addition to several minor neurites left undescribed), most likely related to the anterior sensoria. Similar nerve projections are described in Neodasys chaetonotoideus Remane, 1927 [12, 74], Cephalodasys maximus [37] and Thaidasys tongiorgii Todaro et al. [15] but due to the scarcity of these descriptions, a closer homology cannot yet be stated. Another two pairs of nerves project antero- ventrally from the brain (serotonin-LI-reactive) in D. aspe- tos, one of which may be homologous to the commonly found single pair of serotonin-LI-reactive ventral projec- tions in other gastrotrichs (e.g. Neodasys chaetonotoideus, Fig. 11 Evolution of musculature in Paucitubulatina. Each drawing Dactylopodola or Oregodasys cirratus [12, 36, 38]) (Fig. 12). presents the musculature as organized in a transverse section of the A similar positioned pair of FMRF-amide-LI-reactive trunk with dorsal side pointing at the top. Adapted from [22], with projections is present in Lepidodasys worsaae Hochberg addition of missing longitudinal muscles in the original figure, however, mentioned in the article. The musculature of Neodasys cirritus is modified and Atherton [70] and Xenotrichula [13, 70], and in from [59], and the phylogeny follows the results of the present study Oregodasys cirratus these are expressing both FMRF- (Fig. 1). Possibly homologous muscles are illustrated with similar colors amide-LIR and serotonin-LIR [38], suggesting that the with the dorso-ventral muscles in blue, somatic longitudinal muscles in neurotransmitters of these nerves can vary, and that they green, and splanchnic longitudinal muscles in red. See the discussion for are a general character of Gastrotricha (cf. nervous system further considerations on the different homologies drawing in [2]). Another striking character found in D. aspetos is the paired anterior ventro-median nerve in the anterior topology of our tree (Fig. 1)) or alternatively have been trunk. Short, paired anterior ventro-median nerves are overlooked in previous studies of the sister group Muselli- also found in Thaidasys tongiorgii, Turbanella cf. hya- fer, or the other members of the group A (Fig. 1), Xenotri- lina Schultze [75], and extending the entire body length chulidae. Ruppert [18] also discusses the presence of in Oregodasys cirratus [15, 38, 39]. However, the exact discrete glands opening in the mouth of Chaetonotus and connection to other nerves and their extension differs Musellifer, possibly homologous to the anterior pharyngeal from those of D. aspetos. Moreover, studies on Neodasys glands here described for D. aspetos. chaetonotoideus and on the closely related Xenotrichula Herein is further revealed a presently undescribed [12, 13] did not find similar paired anterior ventro- pharyngeal canal system within the musculature, median nerves and we therefore consider the ventro- Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 25 of 29

Fig. 12 Evolution of the serotonin-LI-reactive nervous system in Chaetonotida. Schematic presentation of dorsal view, anterior end on top, posterior end on bottom. Turbanella ambronensis adapted from [71], Neodasys chaetonotoideus adapted from [12], and Xenotrichula intermedia and X. velox adapted from [13]. If CLSM pictures are available for representatives of the Group B [77], no formal descriptions exist for this group. The phylogeny follows the results of the present study (Fig. 1). See the discussion for further considerations on the different homologies, and variation between the different immunostainings

median nerves in D. aspetos a convergence related to the Ganglia and perikarya Several immunoreactive peri- different ciliation of this species. karya can be compared to other gastrotrichs, mostly The paired short dorso-lateral nerves in D. aspetos (hdpn, Neodasys and Xenotrichula. However, immunoreactivity Figs. 6a, h and 9a) are similar in position and extension to of the perikarya is quite variable, and only a fraction the paired dorsal nerves described in the distantly related of the brain cells are immunoreactive. Macrodasyida C. maximus [37] as well as the dorsal Only five pairs of serotonin-LI-reactive perikarya are pharyngeal fibers found in the phylogenetically close Xeno- found in the brain of Diuronotus aspetos, situated trichula [13] (and Fig. 1, with Muselliferidae sister group of postero-laterally to the neuropil. They comprise two Xenotrichulidae), of which the latter at least seems to be dorsal pairs of perikarya, supplying the neuropil, and a homologous to the dorsal nerves of D. aspetos. ventral pair of para-pharyngeal clusters (spgg, Figs. 7b-e Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 26 of 29

and 9b) with three perikarya each, supplying the ventro- In a position similar to the post-pharyngeal ganglion lateral nerve cords. The closely related Xenotrichula of D. aspetos, two pairs of FMRF-amide-LI-reactive (no does not possess a serotonin-LI-reactive equivalent to serotonin-LIR) perikarya are described supplying the the ventral clusters, but possesses four dorsal pairs of ventro-lateral nerve cord in Xenotrichula [13]. Between serotonin-LI-reactive perikarya [13], two of which are these two pairs, two short transverse FMRF-amide-LI- likely homologous to the two dorsal pairs found in D. reactive neurites almost constitute a commissure similar aspetos (Fig. 12). Neodasys chaetonotoideus possesses to the one of D. aspetos, suggesting that the posterior- three dorso-lateral serotonin-LI-reactive perikarya, most pair of perikarya in Xenotrichula is homologous to which have similar positions and connection to the the ganglia found in D. aspetos. neuropil than the dorsal serotonin-LI-reactive peri- An anal pair of serotonin-LI-reactive perikarya con- karya of D. aspetos. Moreover, N. chaetonotoideus tained in the anal ganglion is found in D. aspetos as well possesses a similar paired cluster of para-pharyngeal as a posterior commissure, similar to what is described serotonin-LI-reactive perikarya, associated to the for Xenotrichula and Neodasys chaetonotoideus [12, 13]. ventro-lateral nerve cords. This suggests that N. chae- Yet, no equivalent is found in any Macrodasyida (Fig. 12), tonotoideus and D. aspetos might share some plesio- confirming that it is either a synapomorphy of Chae- morphic traits of their serotonin-LI-reactive nervous tonotida or Gastrotricha [2], depending of the phylo- system, whereas Xenotrichula represents a derived genetic position of Neodasys. Herein observations condition (Fig. 12). Depending on the position of show that the anal ganglion consists of several cells Neodasys the para-pharyngeal cluster could be either in D. aspetos, contrary to other Chaetonotida [77]. a synapomorphy of Chaetonotida or Gastrotricha. In Moreover, we describe an additional pre-anal commis- the Macrodasyida investigated to date, the serotonin- sure, originating at the anal ganglion, only revealed LI-reactive brain is generally simpler than in Chaeto- by acetylated α-tubulin-LIR and hitherto not found in notida, comprising only one dorsal commissure and other gastrotrichs. one pair of dorso-lateral perikarya [15, 38, 71] (some- times two [76]) (Fig. 12), although additional serotonin- Brain commissures Diuronotus aspetos does not show a LI-reactive perikarya can be found in Dactylopodola commissure situated directly ventrally to the main brain [36], and Paradasys subterraneus Remane [57] (per- neuropil contrary to most Gastrotricha documented, in- sonal observations). cluding Chaetonotida (e.g. [12, 13, 15, 39]). This character The FMRF-amide-LI-reactive perikarya of the brain of was central in previous discussions on a possible close re- D. aspetos are numerous (at least 16 paired and two un- lationship between Cycloneuralia and Gastrotricha (e.g. paired of various intensity of the immunoreactivity) and [36, 39, 78, 79]), rejected today (e.g. [3, 80]) since, recent surround the brain neuropil dorsally, ventrally and lat- interpretations of the brain of Gastrotricha show that it is erally. Due to the high number and variation of FMRF- not truly circular [80]. In D. aspetos, the anterior nerve amide-LIR of the brain in Gastrotricha, we limit our ring is associated to the brain and its ventral portion comparison of D. aspetos to the closely related Xenotri- resembles the ventral brain commissure of other Gastro- chula [13]. Homologies of the perikarya depend on tricha, although being more anterior (Fig. 12). Further- whether the anterior dorsal and ventral FMRF-amide-LI- more, Xenotrichula possesses one ventral FMRF-amide- reactive commissures in Xenotrichula are homologous LI-reactive commissure anterior of the brain [13]. If the to the anterior nerve ring of D. aspetos. If so, the two FMRF-amide-LI-reactive anterior commissure of the brain dorso-median FMRF-amide-LI-reactive perikarya found and ventral commissure of Xenotrichula are continuous, it connected to the anterior dorsal commissure in Xenotri- can be speculated that Xenotrichula also possesses an an- chula, may be homologous to the two dorso-median terior nerve ring, and therefore this arrangement might be found in D. aspetos. The two additional described paired a synapomorphy of the group A (Fig. 1). lateral and ventral FMRF-amide-LI-reactive perikarya in Xenotrichula are difficult to homologize with those of D. Ventral ciliation aspetos. However, one pair of undescribed ventral The main difference from the original description is that FMRF-amide-LI-reactive perikarya is found laterally on the head ventral ciliation forms two medially separated the ventral commissure of Xenotrichula (Figure 4h, [13]) ciliated areas in Diuronotus aspetos. Furthermore, a more and is possibly homologous to the ventral perikarya of detailed pattern has been deduced, showing the relevance the FMRF-amide-LI-reactive nerve ring in D. aspetos. Fi- of CLSM for determining ciliary arrangement [81–83] nally, one of the cells of the FMRF-amide-LI-reactive (but see also Kerbl et al., in prep; Bekkouche and Worsaae, dorso-posterior cluster of the brain of D. aspetos may be in prep, respectively on Dinophilidae (Annelida) and homologous to the single pair of perikarya found in Micrognathozoa). This also opens the way to a new kind Xenotrichula in the same position. of characters in interstitial animals, which could have a Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 27 of 29

systematics value: the pattern of the multi-ciliated cells. evolution of organ systems within Gastrotricha, and Indeed, preliminary results showing variation in the pat- promise new insights for detailed anatomical studies of tern of the ventral multi-ciliated cells of Thaumastoder- the major organ systems. matidae support this idea (Bekkouche and Worsaae The musculature of D. aspetos presents unique traits for unpublished). Unfortunately, though the description of Paucitubulatina (reduction of the number of longitudinal the general pattern of the ventral ciliation is common in muscles, the addition of dorso-ventral muscles) and previ- Chaetonotida, details are rare. In few cases, more details ously undescribed muscles, showing that the musculature were given, for instance for Neogosseidae (with exact de- is more varied than expected in Paucitubulatina [22]. scription of the ciliary bands [84]). A few studies have de- Although the nervous system of D. aspetos is similar scribed ciliary patches in the head of some Chaetonotidae to other gastrotrichs overall, detailed studies revealed (e.g. [85, 86]), but no precise information on the cells numerous unknown minor components of the gastro- themselves has been given, which is why it is unknown trich nervous system (e.g. one pair of anterior ventro- whether each patch or band is composed of one or several median nerves, one pair of dorso-posterior nerves, two cells. This limitation of light and electron microscopy can pairs of additional ganglia). This indicates that similar be overcome by employment of CLSM, but due to the lack detailed immunohistochemical studies on other species of similar studies, we cannot yet comment on the evolu- may similarly reveal new elements in the diversity of the tion of the fine detailed ciliation pattern of Gastrotricha. gastrotrich nervous system and highlight additional Interestingly, some Paucitubulatina show unpaired cil- homologies. This is supported by the multiple similar- iary patches on the ventral midline of the head, e.g. Hali- ities uncovered from comparison with the immunohisto- chaetonotus atlanticus,Kisielewski[85],Arenotus strixinoi chemistry studies of the closely related Neodasys and Kisielewski [86] or Kijanebalola devestiva Todaro et al. Xenotrichula, suggesting, e.g., homologous brain com- [84], but details are insufficient to hypothesize any hom- missures and perikarya. ology with the ventro-median ciliary patches of D. aspetos. The pharynx displays an intriguing canal system and nerves, so far undescribed in Gastrotricha. Additionally, Protonephridial system investigation of the ventral ciliation reveals details of Until the present study, all three previously studied the cellular arrangement which refines the previous Paucitubulatina were known to possess only one pair of description [17] and may show to be of broader sys- protonephridia (Xenotrichula carolinensis stylensis Mock tematic importance within Gastrotricha. [87], Chaetonotus maximus Ehrenberg, 1831 [41, 88] The many new discoveries in the different organ sys- and Polymerurus nodicaudus (Voigt, 1901) [42, 89]). In tems of Gastrotricha unravel an intriguing hidden diver- this context, Diuronotus aspetos is the only Paucitubula- sity of morphological traits in Gastrotricha. This tina known to have more than one pair of protonephri- immediately stresses the importance of similar studies dia, suggesting, according our phylogeny (Fig. 1), an on additional key lineages of Paucitubulatina such as addition of a pair of protonephridia. However, studies on Musellifer, Draculiciteria, and marine Aspidiophorus,as the protonephridial system of Musellifer are needed to well as freshwater “Chaetonotidae,” in order to comple- confirm if the presence of a single pair of protonephridia ment this picture and address the surprisingly complex has a phylogenetic value or is due to size dependency. evolution of gastrotrich morphology. Indeed, the number of pairs of protonephridia in other Gastrotricha is variable and seems to be roughly size Acknowledgements dependent (e.g. two pairs for the ca. 250 μm long Dacty- The Arctic Station of Qeqertarsuaq, University of Copenhagen provided an lopodola baltica, and 11 pairs for the ca. 1 mm long excellent working platform with cooling container and we are thankful to the crew of the station as well as R/V Porsild. We also thank Reinhardt Mesodasys laticaudatus Remane, 1951 [90, 91]). Møbjerg Kristensen for help with collecting, and Christopher Laumer for his help on mining the transcriptome of Diuronotus aspetos, Michelle Jørgensen Conclusion for her help with the sequencing, Alexandra Kerbl for her help on proofreading the manuscript, and Laetitia Carrive for her help with the The present study is the first detailed anatomical de- phylogenetic analysis. scription of a member of Muselliferidae, and only the second description of the nervous system within the lar- Fundings ger clade Paucitubulitina within Gastrotricha [13]. The The fieldwork on Greenland, the lab cost and the salary of the first author key phylogenetic position of Diuronotus in Gastrotricha, was supported by the Villum foundation (Grant no. 102544). the newly discovered traits of the nervous, muscular and ciliary system, and the comparison to phylogenetically Availability of data and materials related lineages reported here lead to new hypotheses on Not applicable to this study, where the only raw data are the large Olympus confocal scanning stacks, all results of which are presented as 2D images in nervous trait homologies and evolution. Our results pro- the figures, why the original scans are irrelevant to share with the reviewers vide an important step towards understanding the and public (and furthermore of incomprehensible size and format). Bekkouche and Worsaae Zoological Letters (2016) 2:21 Page 28 of 29

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